Regulation of phospholipase D activity

ABSTRACT

Novel inhibitors of polyisoprenyl phosphate signaling regulates phopholipase D activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional PatentApplication No. 60/125,194 filed Mar. 18, 1999, the contents of whichare incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The work leading to this invention was supported in part byNational Institutes of Health (NIH) grants GM-38765, DK-50305 andNHLBI-HL-56383. The U.S. Government therefore may have certain rights inthe invention.

BACKGROUND OF THE INVENTION

[0003] Neutrophil (PMN) activation plays a central role in diverse hostresponses, such as host defense, inflammation and reperfusion injury(Weissmann, G., Smolen, J. E., and Korchak, H. M. (1980) Release ofinflammatory mediators from stimulated neutrophils. N. Engl. J. Med.303, 27-34). In response to inflammatory stimuli, PMN phospholipases areactivated to remodel cell membranes and generate bioactive lipids thatserve as intra- or extracellular mediators in the transduction offunctional responses (Serhan, C. N., Haeggstrom, J. Z., and Leslie, C.C. (1996) Lipid mediator networks in cell signaling: update and impactof cytokines. FASEB J. 10, 1147-1158). Important components ofmicrobicidal and acute inflammatory responses include reactive oxygenspecies and granule enzymes that are targeted to phagocytic vacuoles,but aberrant release of these potentially toxic agents can lead toamplification of inflammation as well as tissue injury and areimplicated in a wide range of diseases (Weiss, S. J. (1989) Tissuedestruction by neutrophils. N. Engl. J. Med. 320, 365-376). To preventan over-exuberant inflammatory response and limit damage to the host,these PMN programs are tightly regulated. The host mediators serving asendogenous anti-inflammatory or protective signals are only recentlybeing appreciated (Serhan, C. N. (1994) Lipoxin biosynthesis and itsimpact in inflammatory and vascular events. Biochim. Biophys. Acta 1212,1-25).

SUMMARY OF THE INVENTION

[0004] The present invention pertains to methods for modulating adisease or condition associated with phospholipase D (PLD) activity. Themethods include administration to a subject, an effective anti-PLDamount of a lipoxin analog having the formula described infra, such thatthe PLD initiated activity is modulated.

[0005] The present invention also pertains to methods for treatingphosphlipase D (PLD) activity in a subject. The methods includeadministration of an effective anti-PLD amount of a lipoxin analogdescribed infra, such that PLD initiated activity is treated.

[0006] The present invention further pertains to methods for modulatinga disease or condition associated with phospholipase D (PLD) initiatedgeneration of superoxide or degranulation activity in a subject. Themethods include, administration of an effective anti-PLD amount of alipoxin analog described infra, such that a disease or conditionassociated with initiated by PLD generation of superoxide ordegranulation activity, is modulated.

[0007] The present invention further relates to methods for treatingphospholipase D (PLD) initiated superoxide generation or degranulationactivity in a subject. The methods include administration of aneffective anti-PLD amount of a lipoxin analog described infra, such thatPLD initiated superoxide generation or degranulation activity istreated.

[0008] In preferred embodiments, the methods of the invention areperformed in vitro or in vivo.

[0009] In another aspect, the present invention is directed to apackaged pharmaceutical composition for treating the activity orconditions listed above in a subject. The packaged pharmaceuticalcomposition includes a container holding a therapeutically effectiveamount of at least one lipoxin compound having one of the formulaedescribed infra and instructions for using the lipoxin compound fortreating the activity or condition in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The invention will be more fully understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

[0011]FIG. 1 shows that LTB₄ rapidly remodels PSDP in human PMN:biosynthetic switch by an aspirin-triggered LXA₄ analog. Panel A: Schemefor aspirin-triggered 15-epi-LXA₄ biosynthesis and structure of thestable analog, 15-epi-16-para-fluoro-phenoxy-LXA₄-methyl ester(15-epi-LXa) (left), and hypothetical scheme for PIPP signaling (right).PMN were labeled with [γ-³²P]-ATP and incubated (12.5×10⁶ ml⁻¹, 37° C.)with LTB₄ (, 100 nM), 15-epi-LXa (♦, 100 nM), vehicle (□, 0.1% ethanol)or 15-epi-LXa (100 nM, 5 min) followed by LTB₄ (▴, 100 nM).Non-saponifiable lipids were extracted and separated by TLC, and[³²P]-incorporation was quantitated by phosphoimaging (see Methods).Values are densitometric measurements. Panel B reports a representativetime course (n=5), and Panel C shows the change (mean± S.E.) at 60seconds. *P<0.05 by Student's t-test.

[0012]FIG. 2 demonstrates 15-epi-LXA₄ analog inhibits LTB₄-stimulatedPLD activity and superoxide anion generation. Cell lysates (2−5×10⁶cells, 90-130 μg protein) were prepared from the same aliquots of PMNused to determine PSDP (see FIG. 1 & Methods), warmed to 37° C. andexposed to PC (2 mM in 50 mM Tris-HCl, pH 7.5, plus 30 mM CaCl₂).Reactions were terminated at 30 sec intervals and choline release wasquantitated (26). Values in Panel A are representative (n=5, d=4) of theimpact of 15-epi-LXa on choline release, and Panel B shows the change at60 seconds (mean± S.E.). Superoxide anion generation by freshly isolatedhuman PMN was determined (10 min, 37° C.) for LTB₄ (100 nM), 15-epi-LXa(100 nM), and increasing concentrations of 15-epi-LXa (1-100 nM, 5 min,37° C.) followed by LTB₄ (100 nM, 10 min, 37° C.). Values reported inPanel C are the mean± S.E. for n=3 separate PMN donors. *P<0.05 byStudent's t-test.

[0013]FIG. 3 demonstrates PSDP inhibits phospholipase D. Purified PLD (3units EC 3.1.4.4./125μl ) was warmed (3 min, 30° C.) and exposed to PSDP(10-1000 nM, 5 min, 30° C.) or vehicle (0.04% ethanol final conc.)followed by PC (0.5-5 mM) in 50 mM Tris-HCL (pH 7.5) plus 30 mM CaCl₂.Reactions were terminated at 30 sec intervals and choline releasequantitated as in FIG. 2 legend. Values represent the mean for n>4 forreactions in the absence of PSDP (, r²=0.963) and the mean for n>3 withPSDP (

, r²=0.995, 0.971 and 0.953 for 10, 100 and 1000 nM, respectively). CSChem3D Pro software (CambridgeSoft Corp., Cambridge, Mass.) was used tocalculate an energy minimized model of PSDP (inset).

DETAILED DESCRIPTION OF THE INVENTION

[0014] The features and other details of the invention will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

[0015] It is of wide interest to understand how opposing extracellularsignals (positive or negative) are translated into intracellularsignaling events. Receptor-ligand interactions initiate the generationof bioactive lipids by human neutrophils (PMN) that serve as signals toorchestrate cellular responses important in host defense andinflammation. A novel polyisoprenyl phosphate (PIPP) signaling pathwaywas identified and it was found that one of its components, presqualenediphosphate (PSDP), is a potent negative intracellular signal in PMNthat regulates superoxide anion generation by several stimuli includingphosphatidic acid (Levy et al. (1998) Nature. 389, 985-990). It wasdetermined intracellular PIPP signaling by autacoids with opposingactions on PMN—leukotriene B₄ (LTB₄), a potent chemoattractant, andlipoxin A₄ (LXA₄), a “stop signal” for recruitment. LTB₄ receptoractivation initiated a rapid decrease in PSDP levels concurrent withactivation of PLD and cellular responses. In sharp contrast, activationof the LXA₄ receptor reversed LTB₄-initiated PSDP remodeling leading toan accumulation of PSDP and potent inhibition of both PLD and superoxideanion generation. Thus, an inverse relationship was established for PSDPlevels and PLD activity with two PMN ligands that evoke opposingresponses. In addition, PSDP directly inhibited both isolated humanrecombinant (Ki=6 nM) and plant (Ki=20 nM) PLD. Together, these findingslink PIPP remodeling to intracellular regulation of PMN function andsuggest a role for PIPPs as lipid repressors in signal transduction, anovel mechanism that may also explain aspirin's suppressive actions invivo in cell signaling.

[0016] Bioactive lipids are rapidly generated by activation of cellsurface receptors that carry either specific positive or negativesignals to modulate cellular responses. This is exemplified by therelated eicosanoids, leukotriene B₄ (LTB₄), a potent chemoattractant(Borgeat, P., and Naccache, P. H. (1990) Biosynthesis and biologicalactivity of leukotriene B₄ . Clin. Biochem. 23, 459-468), and lipoxin A₄(LXA₄), an endogenous “stop signal” for PMN recruitment (Serhan, C. N.(1994) Lipoxin biosynthesis and its impact in inflammatory and vascularevents. Biochim. Biophys. Acta 1212, 1-25). LTB₄ and LXA₄ interact withhighly specific and distinct G protein-coupled membrane receptors(Yokomizo, T., Izumi, T., Chang, K., Takuwa, T., and Shimizu, T. (1997)A G-protein-coupled receptor for leukotriene B₄ that mediateschemotaxis. Nature 387, 620-624; Fiore, S., Romano, M., Reardon, E. M.,and Serhan, C. N. (1993) Induction of functional lipoxin A₄ receptors inHL-60 cells. Blood 81, 3395-3403). They each evoke opposing PMNresponses, including LXA₄ inhibition of LTB₄-initiated chemotaxis,adhesion and transmigration (Serhan, C. N. (1994) Lipoxin biosynthesisand its impact in inflammatory and vascular events. Biochim. Biophys.Acta 1212, 1-25).

[0017] Abbreviations used throughout this application include: COX,cyclooxygenase; 15-epi-LX, 15-epimer lipoxin; 15-epi-LXa,15-epi-16-para-fluoro-phenoxy LXA₄-methyl ester; FDP, farnesyldiphosphate; GST, glutathione-S-transferase; LTB₄, leukotriene B₄; LO,lipoxygenase; PA, phosphatidic acid; PC, phosphatidychloine; cPLD,cabbage phospholipase D; PIPP, polyisoprenyl phosphate; PMN,polymorphonuclear leukocytes; PSDP, presqualene diphosphate; PSMP,presqualene monophosphate; Sf9, Spodoptera frugiperda; TLC, thin-layerchromatography.

[0018] Aspirin is known to affect biosynthesis of lipid mediators and iswidely used clinically for its anti-inflammatory properties. Mechanismsresponsible for aspirin's anti-inflammatory actions remain ofconsiderable interest. In particular, new “super-aspirins” are soughtthat spare the gastrointestinal tract and do not possess the deleteriousside effects of steroids (Isakson, P., Seibert, K., Masferrer, J.,Salvemini, D., Lee, L., and Needleman, P. (1995) Discovery of a betteraspirin. Advances in Prostaglandin, Thromboxane & Leukotriene Research23, 49-54). In one aspect it has been that, in addition to inhibitingprostanoid formation, aspirin triggers the endogenous generation ofnovel carbon 15 epimers of LX by transcellular routes (see FIG. 1A)during inflammation in vivo (e.g., between tissue resident cells andinfiltrating leukocytes) (Chiang, N., Takano, T., Clish, C. B., Petasis,N. A., Tai, H.-H., and Serhan, C. N. (1998) Aspirin-triggered15-epi-lipoxin A₄ (ATL) generation by human leukocytes and murineperitonitis exudates: development of a specific 15-epi-LXA₄ ELISA. J.Pharmacol Exper. Ther. 287, 779-790). These aspirin-triggered lipoxins(15-epi-LX) are even more potent than the native LX as inhibitors of PMNresponses, in part because they are active longer (Serhan, C. N.,Maddox, J. F., Petasis, N. A., Akritopoulou-Zanze, I., Papayianni, A.,Brady, H. R., Colgan, S. P., and Madara, J. L. (1995) Design of lipoxinA₄ stable analogs that block transmigration and adhesion of humanneutrophils. Biochemistry 34, 14609-14615). PMN inhibition by LX and15-epi-LX is evoked by specific receptor-activation of “inhibitory”signals and not via direct receptor level antagonism at LTB₄ receptors(Takano, T., Fiore, S., Maddox, J. F., Brady, H. R., Petasis, N. A., andSerhan, C. N. (1997) Aspirin-triggered 15-epi-lipoxin A₄ (LXA₄) and LXA₄Stable analogues are potent inhibitors of acute inflammation: Evidencefor anti-inflammatory receptors. J. Exp. Med. 185, 1693-1704). Moreover,interest in the regulation of the LTB₄ receptor is heightened by therecent finding that LTB₄ receptors also serve as novel HIV-1 coreceptors(Owman, C., Garzino-Demo, A., Cocchi, F., Popovic, M., Sabirsh, A., andGallo, R. (1998) The leukotriene B₄ receptor functions as a novel typeof coreceptor mediating entry of primary HIV-1 isolates intoCD4-positive cells. Proc. Natl. Acad. Sci. 95, 9530-9534).

[0019] Despite ˜100 years of use, complete knowledge of aspirin'stherapeutic impact is still evolving with many newly discovered clinicalutilities (Marcus, A. J. (1995) Aspirin as prophylaxis againstcolorectal cancer. N. Engl. J. Med. 333, 656-658). Regular ingestion ofaspirin decreases the incidence of myocardial infarction, colorectalcarcinoma and Alzheimer's disease (Vainio, H., and Morgan, G. (1997)Aspirin for the second hundred years: new uses for an old drug.Pharmacol Toxicol 81, 151-152), but side effects from aspirin, such asgastrointestinal ulceration, can limit its use. The recent discovery ofa second isoform of cyclooxygenase (COX) that is induced duringinflammation has led to a search for “super-aspirins” that canselectively inhibit COX-2 without disrupting the protective constitutivefunctions of COX-1 (Isakson, P., Seibert, K., Masferrer, J., Salvemini,D., Lee, L., and Needleman, P. (1995) Discovery of a better aspirin.Advances in Prostaglandin, Thromboxane & Leukotriene Research 23, 49-54;Herschman, H. R. (1998) Recent progress in the cellular and molecularbiology of prostaglandin synthesis. Trends in Cardiovasc. Med. 8,145-150). Of particular interest in this regard, 15-epi-LX, whichinhibit PMN migration, are endogenous products of aspirin's acetylatingability that may underly some of the salutary benefits of aspirin. BothLX and 15-epi-LX stable analogs were prepared, which like 15-epi-LXA₄,act via the LXA₄ receptor (Serhan, C. N., Maddox, J. F., Petasis, N. A.,Akritopoulou-Zanze, I., Papayianni, A., Brady, H. R., Colgan, S. P., andMadara, J. L. (1995) Design of lipoxin A₄ stable analogs that blocktransmigration and adhesion of human neutrophils. Biochemistry 34,14609-14615; Takano, T., Fiore, S., Maddox, J. F., Brady, H. R.,Petasis, N. A., and Serhan, C. N. (1997) Aspirin-triggered15-epi-lipoxin A₄ (LXA₄) and LXA₄ Stable analogues are potent inhibitorsof acute inflammation: Evidence for anti-inflammatory receptors. J. Exp.Med. 185, 1693-1704). Suitable methods of preparation of lipoxincompounds can also be found, for example, in U.S. Pat. Nos. 5,411,951,5,648,512, 5,650,435 and 5,750,354, incorporated herein by reference.For example, 15-epi-16-para-fluoro-phenoxy-lipoxin A₄-methyl ester(15-epi-LXa) is a synthetic analog of 15-epi-LXA₄ (FIG. 1A, bottom left)that not only resists rapid inactivation but acts topically to inhibitPMN infiltration and vascular permeability in mouse ear skininflammation (Takano, T., Clish, C. B., Gronert, K., Petasis, N., andSerhan, C. N. (1998) Neutrophil-mediated changes in vascularpermeability are inhibited by topical application of aspirin-triggered15-epi-lipoxin A₄ and novel lipoxin B₄ stable analogues. J. Clin.Invest. 101, 819-826).

[0020] Elucidation of signaling pathway(s) responsible forreceptor-operated blockage of PMN responses is of interest. Signalingvia phospholipase D (PLD) plays a pivotal role in mounting cellularresponses. Within seconds of exposure to ligands, PLD hydrolyzesmembrane phosphatidylcholine (PC) to generate phosphatidic acid(PA)(Billah, M. M., Eckel, S., Mullmann, T. J., Egan, R. W., and Siegel,M. I. (1989) Phosphatidylcholine hydrolysis by phospholipase Ddetermines phosphatidate and diglyceride levels in chemotacticpeptide-stimulated human neutrophils. Involvement of phsophatidatephosphohydrolase in signal transduction. J. Biol. Chem. 264,17069-17077). Formation of PA temporally antecedes functional responses,including vesicle secretion and assembly of the NADPH oxidase (Wakelam,M. J. O., Martin, A., Hodgkin, M. N., Brown, F., Pettit, T. R., Cross,M. J., De Takats, P. G., and Reynolds, J. L. (1997) Role and regulationof phospholipase D activity in normal and cancer cells. Advances inEnzyme Regulation 37, 29-34; Olson, S. C., and Lambeth, J. D. (1996)Biochemistry and cell biology of phospholipase D in human neutrophils.Chem. Phys. Lipids 80, 3-19). Several isozymes of PLD1 and PLD2 werecloned and characterized (Steed, P. M., Clark, K. L., Boyar, W. C., andLasala, D. J. (1998) Characterization of human PLD2 and the analysis ofPLD isoform splice variants. FASEB J. 12, 1309-1317), with PLD1bidentified as a prominent isoform in human granulocytes (Martin, A.,Saqib, K. M., Hodgkin, M. N., Brown, F. D., Pettit, T. R., Armstrong,S., and Wakelam, M. J. O. (1997) Role and regulation of phospholipase Dsignalling. Biochem. Soc. Trans. 25, 1157-1160). The complete DNA andamino acid sequences for human PLD is disclosed in Hammond et al. (1995)J. Biol. Chem. 270: 29640-29643, and Hammond et al. (1997) J. Biol.Chem. 272: 3860-3868, the entire contents of which are incorporatedherein by reference, and can also be found at GenBank Accession Nos. NM002662 and U38545.

[0021] Recently, a novel polyisoprenyl phosphate (PIPP) signalingpathway was identified (FIG. 1A) and found that, in PMN, presqualenediphosphate (PSDP) carries biological activity and serves as a negativeintracellular signal that prevents superoxide anion generation byseveral stimuli including PA (Levy, B. D., Petasis, N. A., and Serhan,C. N. (1997) Polyisoprenyl phosphates in intracellular signalling.Nature 389, 985-989). Because PLD activation is linked to superoxideanion generation (Agwu, D. E., McPhail, L. C., Sozzani, S., Bass, D. A.,and McCall, C. E. (1991) Phosphatidic acid as a second messenger inhuman polymorphonuclear leukocytes. Effects on activation of NADPHoxidase. J. Clin. Invest. 88, 531-539), it was determined that PIPPsignaling also modulates phospholipase activity critical to globalcellular activation. It was found that (i) that LTB₄ receptor activationrapidly degrades PSDP, a key component of PIPP signaling, that isreversed by a LXA₄ receptor agonist, (ii) that an aspirin-triggered15-epi-LXA₄ stable analog potently inhibits LTB₄-initiated PLDactivation and superoxide anion generation, and (iii) that PSDP directlyinhibits both human recombinant and plant PLD. These findings provideevidence for receptor-initiated PIPP remodeling as a regulatorysignaling pathway.

[0022] The present invention pertains to methods for modulating adisease or condition associated with phospholipase D (PLD) activity. Themethods include administration to a subject, an effective anti-PLDamount of a lipoxin analog having the formula described infra, such thatthe PLD initiated activity is modulated.

[0023] The present invention also pertains to methods for treatingphosphlipase D (PLD) activity in a subject. The methods includeadministration of an effective anti-PLD amount of a lipoxin analogdescribed infra, such that PLD initiated activity is treated.

[0024] The present invention further pertains to methods for modulatinga disease or condition associated with phospholipase D (PLD) initiatedgeneration of superoxide or degranulation activity in a subject. Themethods include, administration of an effective anti-PLD amount of alipoxin analog described infra, such that a disease or conditionassociated with initiated by PLD generation of superoxide ordegranulation activity, is modulated.

[0025] The present invention further relates to methods for treatingphospholipase D (PLD) initiated superoxide generation or degranulationactivity in a subject. The methods include administration of aneffective anti-PLD amount of a lipoxin analog described infra, such thatPLD initiated superoxide generation or degranulation activity istreated.

[0026] In preferred embodiments, the methods of the invention areperformed in vitro or in vivo.

[0027] In another aspect, the present invention is directed to apackaged pharmaceutical composition for treating the activity orconditions listed above in a subject. The packaged pharmaceuticalcomposition includes a container holding a therapeutically effectiveamount of at least one lipoxin compound having one of the formulaedescribed infra and instructions for using the lipoxin compound fortreating the activity or condition in the subject.

[0028] In one embodiment, compounds useful in the invention have theformula (I)

[0029] wherein X is R₁, OR₁, or SR₁; wherein R₁ is

[0030] (i) a hydrogen atom;

[0031] (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may bestraight chain or branched;

[0032] (iii) a cycloalkyl of 3 to 10 carbon atoms;

[0033] (iv) an aralkyl of 7 to 12 carbon atoms;

[0034] (v) phenyl;

[0035] (vi) substituted phenyl

[0036]  wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are eachindependently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogenatom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms,inclusive, which may be a straight chain or branched, and hydroxyl;

[0037] (vii) a detectable label molecule; or

[0038] (viii) a straight or branched chain alkenyl of 2 to 8 carbonatoms, inclusive;

[0039] wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then Xis absent;

[0040] wherein Q₃ and Q₄ are each independently O, S or NH;

[0041] wherein one of R₂ and R₃ is a hydrogen atom and the other is

[0042] (a) H;

[0043] (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched;

[0044] (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;

[0045] (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may bestraight chain or branched; or

[0046] (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) isalkylene of 0 to 6 carbons atoms, inclusive, which may be straight chainor branched and wherein R_(b) is alkyl of 0 to 8 carbon atoms,inclusive, which may be straight chain or branched, provided when R_(b)is O, then R_(b) is a hydrogen atom;

[0047] wherein R₄ is

[0048] (a) H;

[0049] (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched;

[0050] wherein R₅ is

[0051] wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are eachindependently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogenatom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms,inclusive, which may be a straight chain or branched, and hydroxyl or asubstituted or unsubstituted, branched or unbranched alkyl group;

[0052] wherein Y₁ is —OH, methyl, —SH, an alkyl of 2 to 4 carbon atoms,inclusive, straight chain or branched, an alkoxy of 1 to 4 carbon atoms,inclusive, or CH_(a)Z_(b) where a+b=3, a =0 to 3, b=0 to 3 and Z iscyano, nitro or a halogen;

[0053] wherein R₆ is

[0054] (a) H;

[0055] (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chainor branched;

[0056] wherein T is O or S, and pharmaceutically acceptable saltsthereof.

[0057] In another embodiment, compounds useful in the invention have theformula (II)

[0058] wherein X is R₁, OR₁, or SR₁;

[0059] wherein R₁ is

[0060] (i) a hydrogen atom;

[0061] (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may bestraight chain or branched;

[0062] (iii) a cycloalkyl of 3 to 10 carbon atoms;

[0063] (iv) an aralkyl of 7 to 12 carbon atoms;

[0064] (v) phenyl;

[0065] (vi) substituted phenyl

[0066]  wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are eachindependently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogenatom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms,inclusive, which may be a straight chain or branched, and hydroxyl;

[0067] (vii) a detectable label molecule; or

[0068] (viii) a straight or branched chain alkenyl of 2 to 8 carbonatoms, inclusive;

[0069] wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then Xis absent;

[0070] wherein one of R₂ and R₃ is a hydrogen atom and the other is

[0071] (a) H;

[0072] (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched;

[0073] (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;

[0074] (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may bestraight chain or branched; or

[0075] (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) isalkylene of 0 to 6 carbons atoms, inclusive, which may be straight chainor branched and wherein R_(b) is alkyl of 0 to 8 carbon atoms,inclusive, which may be straight chain or branched, provided when R_(b)is 0, then R_(b) is a hydrogen atom;

[0076] wherein R₄ is

[0077] (a) H;

[0078] (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched;

[0079] wherein R₅ is

[0080] wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are eachindependently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogenatom, halogen, methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms,inclusive, which may be a straight chain or branched, and hydroxyl or asubstituted or unsubstituted, branched or unbranched alkyl group;

[0081] wherein Y₁ is —OH, methyl, —SH, an alkyl of 2 to 4 carbon atoms,inclusive, straight chain or branched, an alkoxy of 1 to 4 carbon atoms,inclusive, or CH_(a)Z_(b) where a+b=3, a=0 to 3, b=0 to 3 and Z iscyano, nitro or a halogen;

[0082] wherein R₆ is

[0083] (a) H;

[0084] (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chainor branched;

[0085] wherein T is O or S, and pharmaceutically acceptable saltsthereof.

[0086] The invention is also directed to useful lipoxin compounds havingthe formula

[0087] wherein X is R₁, OR₁, or SR₁;

[0088] wherein R₁ is

[0089] (i) a hydrogen atom;

[0090] (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may bestraight chain or branched;

[0091] (iii) a cycloalkyl of 3 to 10 carbon atoms;

[0092] (iv) an aralkyl of 7 to 12 carbon atoms;

[0093] (v) phenyl;

[0094] (vi) substituted phenyl

[0095]  wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are eachindependently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogenatom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms,inclusive, which may be a straight chain or branched, and hydroxyl;

[0096] (vii) a detectable label molecule; or

[0097] (viii) a straight or branched chain alkenyl of 2 to 8 carbonatoms, inclusive;

[0098] wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then Xis absent;

[0099] wherein one of R₂ and R₃ is a hydrogen atom and the other is

[0100] (a) H;

[0101] (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched;

[0102] (c) a cycloalkyl of 3 to 6 carbon atoms, inclusive;

[0103] (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which may bestraight chain or branched; or

[0104] (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or —S—; wherein R_(a) isalkylene of 0 to 6 carbons atoms, inclusive, which may be straight chainor branched and wherein R_(b) is alkyl of 0 to 8 carbon atoms,inclusive, which may be straight chain or branched, provided when R_(b)is 0, then R_(b) is a hydrogen atom;

[0105] wherein R₄ is

[0106] (a) H;

[0107] (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched;

[0108] wherein R₅ is

[0109]  wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are eachindependently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogenatom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms,inclusive, which may be a straight chain or branched, and hydroxyl or asubstituted or unsubstituted, branched or unbranched alkyl group;

[0110] wherein R₆ is

[0111] (a) H;

[0112] (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chainor branched;

[0113] wherein T is O or S, and pharmaceutically acceptable saltsthereof.

[0114] The invention is further directed to useful lipoxin compoundshaving the formula (IV)

[0115] wherein X is R₁, OR₁, or SR₁;

[0116] wherein R₁ is

[0117] (i) a hydrogen atom;

[0118] (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may bestraight chain or branched;

[0119] (iii) a cycloalkyl of 3 to 10 carbon atoms;

[0120] (iv) an aralkyl of 7 to 12 carbon atoms;

[0121] (v) phenyl;

[0122] (vi) substituted phenyl

[0123]  wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are eachindependently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogenatom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms,inclusive, which may be a straight chain or branched, and hydroxyl;

[0124] (vii) a detectable label molecule; or

[0125] (viii) a straight or branched chain alkenyl of 2 to 8 carbonatoms, inclusive;

[0126] wherein Q₁ is (C═O), SO₂ or (CN), provided when Q₁ is CN, then Xis absent;

[0127] wherein R₄ is

[0128] (a) H;

[0129] (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched;

[0130] wherein R₅ is

[0131]  wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are eachindependently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogenatom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms,inclusive, which may be a straight chain or branched, and hydroxyl or asubstituted or unsubstituted, branched or unbranched alkyl group;

[0132] wherein R₆ is

[0133] (a) H;

[0134] (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chainor branched;

[0135] wherein T is O or S, and pharmaceutically acceptable saltsthereof.

[0136] The invention is further directed to useful lipoxin compoundshaving the formula (V)

[0137] wherein X is R₁, OR₁, or SR₁;

[0138] wherein R₁ is

[0139] (i) a hydrogen atom;

[0140] (ii) an alkyl of 1 to 8 carbons atoms, inclusive, which may bestraight chain or branched;

[0141] (iii) a cycloalkyl of 3 to 10 carbon atoms;

[0142] (iv) an aralkyl of 7 to 12 carbon atoms;

[0143] (v) phenyl;

[0144] (vi) substituted phenyl

[0145]  wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are eachindependently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogenatom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms,inclusive, which may be a straight chain or branched, and hydroxyl;

[0146] (vii) a detectable label molecule; or

[0147] (viii) a straight or branched chain alkenyl of 2 to 8 carbonatoms, inclusive;

[0148] wherein R₄ is

[0149] (a) H;

[0150] (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched;

[0151] wherein R₅ is

[0152]  wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are eachindependently selected from —NO_(2,)—CN, —C(═O)—R₁, —SO₃H, a hydrogenatom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms,inclusive, which may be a straight chain or branched, and hydroxyl or asubstituted or unsubstituted, branched or unbranched alkyl group;

[0153] wherein R₆ is

[0154] (a) H;

[0155] (b) an alkyl from 1 to 4 carbon atoms, inclusive, straight chainor branched; and

[0156] pharmaceutically acceptable salts thereof.

[0157] In preferred embodiments, X is OR₁ wherein R₁ is a hydrogen atom,an alkyl group of 1 to 4 carbon atoms or a pharmaceutically acceptablesalt, Q₁ is C═O, R₂ and R₃, if present, are hydrogen atoms, R₄ is ahydrogen atom or methyl, Q₃ and Q₄, if present, are both O, R₆, ifpresent, is a hydrogen atom, Y₁, if present, is OH, T is O and R₅ is asubstituted phenyl, e.g.,

[0158] wherein Z_(i), Z_(ii), Z_(iii), Z_(iv) and Z_(v) are eachindependently selected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogenatom, halogen, methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms,inclusive, which may be a straight chain or branched, and hydroxyl. Incertain embodiments for R₅, para-fluorophenyl and/or unsubstitutedphenyl are excluded, e.g.,15-epi-16-(para-fluoro)-phenoxy-LXA_(4, 16)-(para-fluoro)-phenoxy-LXA_(4, 15)-epi-16-phenoxy-LXA₄or 16-phenoxy-LXA₄. The compounds encompassed by U.S. Pat. No. 5,441,951are excluded from certain aspects of the present invention.

[0159] In still another aspect, the present invention is directed topharmaceutical compositions including compounds having theabove-described formulae and a pharmaceutically acceptable carrier. Inone embodiment, a preferred compound is

[0160] In a preferred embodiment, the pharmaceutical carrier is not aketone, e.g., acetone.

[0161] In preferred embodiments, Y₁ is a hydroxyl and the carbon bearingthe hydroxyl can have an R or S configuration. In most preferredembodiments, the chiral carbon bearing the hydroxyl group, e.g., Y₁ isdesignated as a 15-epi-lipoxin as is known in the art.

[0162] In certain embodiments the chirality of the carbons bearing theR₂, R₃, Q₃ and Q₄ groups can each independently be either R or S. Inpreferred embodiments, Q₃ and Q₄ have the chiralities shown instructures II, III, IV or V.

[0163] In preferred embodiments, R₄ is a hydrogen. In other preferredembodiments, R₆ is a hydrogen.

[0164] Additionally, R₅ can be a substituted or unsubstituted, branchedor unbranched alkyl group having between 1 and about 6 carbon atoms,preferably between 1 and 4 carbon atoms, most preferably between 1 and3, and preferably one or two carbon atoms. The carbon atoms can havesubstituents which include halogen atoms, hydroxyl groups, or ethergroups.

[0165] The compounds useful in the present invention can be prepared bythe following synthetic scheme:

[0166] wherein X, Q₁, Q₃, Q₄, R₂, R₃, R₄, R₅, R₆, Y₁ and T are asdefined above. Suitable methods known in the art to can be used toproduce each fragment. For example, the acetylenic fragment can beprepared by the methods discussed in Nicolaou, K. C. et al. (1991)Angew. Chem. Int. Ed. Engl. 30:1100; Nicolaou, K. C. et al. (1989) J.Org. Chem. 54:5527; Webber, S.E. et al. (1988) Adv. Exp. Med. Biol.229:61; and U.S. Pat. No. 5,441,951. The second fragment can be preparedby the methods of Raduchel, B. and Vorbruggen, H. (1985) Adv.Prostaglandin Thromboxane Leukotriene Res. 14:263.

[0167] A “lipoxin analog” shall mean a compound which has an “activeregion” that functions like the active region of a “natural lipoxin”,but which has a “metabolic transformation region” that differs fromnatural lipoxin. Lipoxin analogs include compounds which arestructurally similar to a natural lipoxin, compounds which share thesame receptor recognition site, compounds which share the same orsimilar lipoxin metabolic transformation region as lipoxin, andcompounds which are art-recognized as being analogs of lipoxin. Lipoxinanalogs include lipoxin analog metabolites. The compounds disclosedherein may contain one or more centers of asymmetry. Where asymmetriccarbon atoms are present, more than one stereoisomer is possible, andall possible isomeric forms are intended to be included within thestructural representations shown. Optically active (R) and (S) isomersmay be resolved using conventional techniques known to the ordinarilyskilled artisan. The present invention is intended to include thepossible diastereiomers as well as the racemic and optically resolvedisomers.

[0168] The terms “corresponding lipoxin” and “natural lipoxin” refer toa naturally-occurring lipoxin or lipoxin metabolite. Where an analog hasactivity for a lipoxin-specific receptor, the corresponding or naturallipoxin is the normal ligand for that receptor. For example, where ananalog is a LXA₄ specific receptor on differentiated HL-60 cells, thecorresponding lipoxin is LXA₄. Where an analog has activity as anantagonist to another compound (such as a leukotriene), which isantagonized by a naturally-occurring lipoxin, that natural lipoxin isthe corresponding lipoxin.

[0169] “Active region” shall mean the region of a natural lipoxin orlipoxin analog, which is associated with in vivo cellular interactions.The active region may bind the “recognition site” of a cellular lipoxinreceptor or a macromolecule or complex of macromolecules, including anenzyme and its cofactor. Preferred lipoxin A₄ analogs have an activeregion comprising C₅-C₁₅ of natural lipoxin A₄. Preferred lipoxin B₄analogs have an active region comprising C₅-C₁₄ of natural lipoxin B4.

[0170] The term “recognition site” or receptor is art-recognized and isintended to refer generally to a functional macromolecule or complex ofmacromolecules with which certain groups of cellular messengers, such ashormones, leukotrienes, and lipoxins, must first interact before thebiochemical and physiological responses to those messengers areinitiated. As used in this application, a receptor may be isolated, onan intact or permeabilized cell, or in tissue, including an organ. Areceptor may be from or in a living subject, or it may be cloned. Areceptor may normally exist or it may be induced by a disease state, byan injury, or by artificial means. A compound of this invention may bindreversibly, irreversibly, competitively, noncompetitively, oruncompetitively with respect to the natural substrate of a recognitionsite.

[0171] The term “metabolic transformation region” is intended to refergenerally to that portion of a lipoxin, a lipoxin metabolite, or lipoxinanalog including a lipoxin analog metabolite, upon which an enzyme or anenzyme and its cofactor attempts to perform one or more metabolictransformations which that enzyme or enzyme and cofactor normallytransform on lipoxins. The metabolic transformation region may or maynot be susceptible to the transformation. A nonlimiting example of ametabolic transformation region of a lipoxin is a portion of LXA₄ thatincludes the C-13,14 double bond or the C-15 hydroxyl group, or both.

[0172] The term “detectable label molecule” is meant to includefluorescent, phosphorescent, and radiolabeled molecules used to trace,track, or identify the compound or receptor recognition site to whichthe detectable label molecule is bound. The label molecule may bedetected by any of the several methods known in the art.

[0173] The term “labeled lipoxin analog” is further understood toencompass compounds which are labeled with radioactive isotopes, such asbut not limited to tritium (³H), deuterium (²H), carbon (¹⁴C), orotherwise labeled (e.g. fluorescently). The compounds of this inventionmay be labeled or derivatized, for example, for kinetic bindingexperiments, for further elucidating metabolic pathways and enzymaticmechanisms, or for characterization by methods known in the art ofanalytical chemistry.

[0174] The term “inhibits metabolism” means the blocking or reduction ofactivity of an enzyme which metabolizes a native lipoxin. The blockageor reduction may occur by covalent bonding, by irreversible binding, byreversible binding which has a practical effect of irreversible binding,or by any other means which prevents the enzyme from operating in itsusual manner on another lipoxin analog, including a lipoxin analogmetabolite, a lipoxin, or a lipoxin metabolite.

[0175] The term “resists metabolism” is meant to include failing toundergo one or more of the metabolic degradative transformations by atleast one of the enzymes which metabolize lipoxins. Two nonlimitingexamples of LXA₄ analog that resists metabolism are 1) a structure whichcan not be oxidized to the 15-oxo form, and 2) a structure which may beoxidized to the 15-oxo form, but is not susceptible to enzymaticreduction to the 13,14-dihydro form.

[0176] The term “more slowly undergoes metabolism” means having slowerreaction kinetics, or requiring more time for the completion of theseries of metabolic transformations by one or more of the enzymes whichmetabolize lipoxin. A nonlimiting example of a LXA₄ analog which moreslowly undergoes metabolism is a structure which has a higher transitionstate energy for C-15 dehydrogenation than does LXA₄ because the analogis sterically hindered at the C-16.

[0177] The term “tissue” is intended to include intact cells, blood,blood preparations such as plasma and serum, bones, joints, muscles,smooth muscles, and organs.

[0178] The term “halogen” is meant to include fluorine, chlorine,bromine and iodine, or fluoro, chloro, bromo, and iodo. In certainaspects, the compounds of the invention do not include halogenatedcompounds, e.g., fluorinated compounds.

[0179] The term “subject” is intended to include living organismssusceptible to conditions or diseases caused or contributed to byinflammation, inflammatory responses, vasoconstriction, and myeloidsuppression. Examples of subjects include humans, dogs, cats, cows,goats, and mice. The term subject is further intended to includetransgenic species.

[0180] When the compounds of the present invention are administered aspharmaceuticals, to humans and mammals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

[0181] The phrase “pharmaceutically acceptable carrier” as used hereinmeans a pharmaceutically acceptable material, composition or vehicle,such as a liquid or solid filler, diluent, excipient, solvent orencapsulating material, involved in carrying or transporting acompound(s) of the present invention within or to the subject such thatit can perform its intended function. Typically, such compounds arecarried or transported from one organ, or portion of the body, toanother organ, or portion of the body. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not injurious to the patient. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include: sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients, such as cocoa butter andsuppository waxes; oils, such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil; glycols, such aspropylene glycol; polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations.

[0182] In certain embodiment, the compounds of the present invention maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically acceptable salts with pharmaceuticallyacceptable bases. The term “pharmaceutically acceptable salts” in theseinstances refers to the relatively non-toxic, inorganic and organic baseaddition salts of compounds of the present invention. These salts canlikewise be prepared in situ during the final isolation and purificationof the compounds, or by separately reacting the purified compound in itsfree acid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like.

[0183] The term “pharmaceutically acceptable esters” refers to therelatively non-toxic, esterified products of the compounds of thepresent invention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. The term isfurther intended to include lower hydrocarbon groups capable of beingsolvated under physiological conditions, e.g., alkyl esters, methyl,ethyl and propyl esters. In a preferred embodiment, the ester is not amethyl ester (See, for example, Berge et al., supra.).

[0184] Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

[0185] Examples of pharmaceutically acceptable antioxidants include:water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0186] Formulations of the present invention include those suitable forintravenous, oral, nasal, topical, transdermal, buccal, sublingual,rectal, vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect. Generally, out of one hundred per cent,this amount will range from about 1 percent to about ninety-nine percentof active ingredient, preferably from about 5 percent to about 70percent, most preferably from about 10 percent to about 30 per cent.

[0187] Methods of preparing these formulations or compositions includethe step of bringing into association a compound of the presentinvention with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

[0188] Formulations of the invention suitable for oral administrationmay be in the form of capsules, cachets, pills, tablets, lozenges (usinga flavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

[0189] In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

[0190] A tablet may be made by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be preparedusing binder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

[0191] The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

[0192] Liquid dosage forms for oral administration of the compounds ofthe invention include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

[0193] Besides inert diluents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

[0194] Suspensions, in addition to the active compounds, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

[0195] Formulations of the pharmaceutical compositions of the inventionfor rectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

[0196] Formulations of the present invention which are suitable forvaginal administration also include pessaries, tampons, creams, gels,pastes, foams or spray formulations containing such carriers as areknown in the art to be appropriate.

[0197] Dosage forms for the topical or transdermal administration of acompound of this invention include powders, sprays, ointments, pastes,creams, lotions, gels, solutions, patches and inhalants. The activecompound may be mixed under sterile conditions with a pharmaceuticallyacceptable carrier, and with any preservatives, buffers, or propellantswhich may be required.

[0198] The ointments, pastes, creams and gels may contain, in additionto an active compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

[0199] Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

[0200] Transdermal patches have the added advantage of providingcontrolled delivery of a compound of the present invention to the body.Such dosage forms can be made by dissolving or dispersing the compoundin the proper medium. Absorption enhancers can also be used to increasethe flux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe active compound in a polymer matrix or gel.

[0201] Ophthalmic formulations, eye ointments, powders, solutions andthe like, are also contemplated as being within the scope of thisinvention.

[0202] Pharmaceutical compositions of this invention suitable forparenteral administration comprise one or more compounds of theinvention in combination with one or more pharmaceutically acceptablesterile isotonic aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, or sterile powders which may be reconstitutedinto sterile injectable solutions or dispersions just prior to use,which may contain antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

[0203] Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

[0204] These compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

[0205] In some cases, in order to prolong the effect of a drug, it isdesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

[0206] Injectable depot forms are made by forming microencapsulematrices of the subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

[0207] The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Intravenous injection administration is preferred.

[0208] The phrases “parenteral administration” and “administeredparenterally” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrastemalinjection and infusion.

[0209] The phrases “systemic administration,” “administeredsystematically,” “peripheral administration” and “administeredperipherally” as used herein mean the administration of a compound, drugor other material other than directly into the central nervous system,such that it enters the patient's system and, thus, is subject tometabolism and other like processes, for example, subcutaneousadministration.

[0210] These compounds may be administered to humans and other animalsfor therapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

[0211] Regardless of the route of administration selected, the compoundsof the present invention, which may be used in a suitable hydrated form,and/or the pharmaceutical compositions of the present invention, areformulated into pharmaceutically acceptable dosage forms by conventionalmethods known to those of ordinary skill in the art.

[0212] Actual dosage levels of the active ingredients in thepharmaceutical compositions of this invention may be varied so as toobtain an amount of the active ingredient which is effective to achievethe desired therapeutic response for a particular patient, composition,and mode of administration, without being toxic to the patient.

[0213] The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

[0214] A physician or veterinarian having ordinary skill in the art canreadily determine and prescribe the effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

[0215] In general, a suitable daily dose of a compound of the inventionwill be that amount of the compound which is the lowest dose effectiveto produce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous andsubcutaneous doses of the compounds of this invention for a patient,when used for the indicated analgesic effects, will range from about0.0001 to about 100 mg per kilogram of body weight per day, morepreferably from about 0.01 to about 50 mg per kg per day, and still morepreferably from about 0.1 to about 40 mg per kg per day. For example,between about 0.01 microgram and 20 micrograms, between about 20micrograms and 100 micrograms and between about 10 micrograms and 200micrograms of the compounds of the invention are administered per 20grams of subject weight.

[0216] If desired, the effective daily dose of the active compound maybe administered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

[0217] While it is possible for a compound of the present invention tobe administered alone, it is preferable to administer the compound as apharmaceutical composition.

[0218] Methods

[0219] Materials.

[0220] 15-epi-LXa, PSDP and PSMP were prepared by total organicsynthesis and characterized by their physical chemical and biologicalproperties (Takano, T., Clish, C. B., Gronert, K., Petasis, N., andSerhan, C. N. (1998) Neutrophil-mediated changes in vascularpermeability are inhibited by topical application of aspirin-triggered15-epi-lipoxin A₄ and novel lipoxin B₄ stable analogues. J. Clin.Invest. 101, 819-826; Levy, B. D., Petasis, N. A., and Serhan, C. N.(1997) Polyisoprenyl phosphates in intracellular signaling. Nature 389,985-989). LTB₄ was purchased from Cayman Chemical (Ann Arbor, Mich.),cabbage PLD (cPLD), FDP, squalene, lysis buffer reagents and cytochromec were from Sigma Chemical Co. (St. Louis, Mo.), and PC and PA were fromAvanti Polar Lipids (Alabaster, Ala.). The integrity and concentrationof each bioactive lipid was assessed just prior to each series ofexperiments by UV analysis (eicosanoids and analogs) and phosphorusdeterminations (polyisoprenyl phosphates) (Takano, T., Clish, C. B.,Gronert, K., Petasis, N., and Serhan, C. N. (1998) Neutrophil-mediatedchanges in vascular permeability are inhibited by topical application ofaspirin-triggered 15-epi-lipoxin A₄ and novel lipoxin B₄ stableanalogues. J. Clin. Invest. 101, 819-826; Levy, B. D., Petasis, N. A.,and Serhan, C. N. (1997) Polyisoprenyl phosphates in intracellularsignaling. Nature 389, 985-989).

[0221] Human PMN

[0222] Peripheral venous blood (˜180 ml) was obtained by venipuncturefrom healthy volunteers who denied taking any medication for at leasttwo weeks and had given written informed consent to a protocol approvedby Brigham and Women's Hospital's Human Research Committee. PMN wereisolated from whole blood and steady state labeled with [γ-³²P]ATP (40μCiml⁻¹, 90 min, 37° C.) as in (Levy, B. D., Petasis, N. A., and Serhan,C. N. (1997) Polyisoprenyl phosphates in intracellular signalling.Nature 389, 985-989). Labeled PMN were resuspended (20×10⁶ ml⁻¹ PBS with1 mM CaCl₂, pH 7.40) and exposed to LTB₄ (100 nM), 15-epi-LXa (100 nM)or vehicle (0.1% EtOH) for 0 to 300 seconds (37° C.). From eachincubation, aliquots were removed at indicated intervals to determinethe radiolabeling of nonsaponifiable lipids (10−12×10⁶ PMN) and PLDactivity (1−1.25×10⁶ PMN). Materials present in each incubation weresaponified, extracted and separated by TLC with phosphoimaging (model425E and integration software; Molecular Dynamics), which was used forPSDP mass determination as in ref 22.

[0223] Preparation of recombinant human PLD1b.

[0224] Spodoptera frugiperda (Sf9) cells were cultured in suspension at2×10⁵ to 2×10⁶ cells/ml TC100 medium supplemented with 10% fetal calfserum (Gibco). A cDNA encoding human PLD1b (cloned from placentaltissue) was inserted into the transfer vector pACGHLT (Pharmingen)downstream of, and in frame with, vector sequences encodingglutathione-S-transferase (GST), hexahistidine, a protein kinase Aphosphorylation site and a thrombin cleavage site. The GST-hPLD1bconstruct was cotransfected into Sf9 cells with linearized,polyhedrin-minus (PH-), AcMNPV DNA, Bac-N-Blue, according to thesupplier's instructions (Invitrogen). Homologous recombination betweenlinearized virus and the transfer vector restored the function ofessential viral gene ORF1629 to yield infectious, recombinant virus.After two rounds of plaque-purification, recombinant virus was amplifiedby large scale infections of Sf9 cells until a titer of 8×10⁷ pfu/ml wasobtained. To generate GST-hPLD1b, 500 ml of Sf9 cells at 2×10⁶ cells/mlwere infected with virus at a multiplicity of infection of 10:1. Cellswere harvested 72 hours post-infection, lysed and the expressedGST-hPLD1b purified on glutathione agarose beads, according tosupplier's instructions (Pharmingen). The purified recombinant proteinwas identified by immunoreactivity with goat anti-GST pAb (AmershamPharmacia Biotech) and rabbit pAb raised against the PLD consensuspeptide sequence GSANIN (gift of P. Parker, ICRF, London, UK), and byactivity in an in vitro PLD assay (24).

[0225] PLD Activity and Superoxide Anion Generation.

[0226] Lysates were generated from cells at rest or after exposure toagonist using a lysis buffer comprised of 0.1 M Hepes (pH 7.4), 0.7 mMsodium orthovanadate, 10 μM p-nitrophenylphosphate, 10 mM EGTA, 5.5%triton X-100, 0.5 M β-glycerophosphate, 10 mMphenylmethylsulfonylfluoride, 0.1 mM ammonium molybdate, 12 mM DFP, 5μgml⁻¹ leupeptin, 2 μgml⁻¹ aprotinin and 7 μgml⁻¹ pepstatin A (as in ref25) and utilized for bioassay.

[0227] PMN lysates (90-130 μg protein), purified phospholipase D (3-30units) (EC 3.1.4.4., Sigma Chemical Co.) or recombinant hPLD1b werewarmed (37° C. for mammalian enzyme and 30° C. for cabbage, 3 min) andexposed to PSDP, PSMP or FDP (10-1000 μM, 5 min, 37° C. or 30° C.)followed by PC (0.5 to 5 mM) in Tris-HCl (50 mM, pH 7.5) with CaCl₂ (30mM). Reactions were terminated at 30 second intervals (0-90 seconds)with Tris-HCl (1 M) plus EDTA (50 mM). Choline release was quantitatedas in ref 26.

[0228] Freshly isolated human PMN (1-3×10⁶ PMN/ml HBSS+1.6 mM CaCl₂)were incubated (5 min, 37° C.) in the presence of 15-epi-LXa (1-100 nM)or vehicle (0.1% ethanol), and then exposed (10 min) to LTB₄ (100 nM) inthe presence of cytochrome c (7 mg/ml). Superoxide anion generation wasdetermined as in ref 22.

[0229] Statistical Analysis.

[0230] Results are expressed as the mean± S.E. and statisticalsignificance was evaluated using the Student's test.

[0231] Results

[0232] Leukotriene B₄ Stimulates Rapid Remodeling of PIPP: Degradationof PSDP.

[0233] Leukotriene B₄ interacts with its receptor to rapidly activatephospholipases and signal cellular responses (Yokomizo, T., Izumi, T.,Chang, K., Takuwa, T., and Shimizu, T. (1997) A G-protein-coupledreceptor for leukotriene B₄ that mediates chemotaxis. Nature 387,620-624). To determine if LTB₄ receptor activation lead to remodeling ofPIPP and specifically PSDP, cellular phosphate pools were steady statelabeled with [γ-³²P]-ATP (see Methods) and exposed to either LTB₄ (100nM) or vehicle (0.1% ethanol) alone.

[0234] Aliquots were removed at timed intervals from 0 to 300 sec (37°C.) and non-saponifiable phosphorylated lipids were isolated andquantitated by phosphoimager for [³²P] incorporation. PSDP levels inunstimulated PMN are ˜1.7 nmoles/10⁷ PMN (˜50 nM) (Levy, B. D., Petasis,N. A., and Serhan, C. N. (1997) Polyisoprenyl phosphates inintracellular signalling. Nature 389, 985-989). PSDP and presqualenemonophosphate (PSMP), but not farnesyl diphosphate (FDP), incorporated[³²P] from ATP, consistent with recent evidence (Levy, B. D., Petasis,N. A., and Serhan, C. N. (1997) Polyisoprenyl phosphates inintracellular signalling. Nature 389, 985-989). LTB₄ initiated a rapid(evident within 30 sec) (FIG. 1B) and statistically significant decreasein [³²P]-PSDP (28%) within 60 sec (FIG. 1C). Within the ensuing 270 sec,[³²P]-PSDP levels returned to baseline amounts (t=0). Changes in[³²P]-PSDP after LTB₄ receptor activation reflected changes in PSDPmass. These results confirmed that PSDP was present in PMN (Levy, B. D.,Petasis, N. A., and Serhan, C. N. (1997) Polyisoprenyl phosphates inintracellular signalling. Nature 389, 985-989) and indicated that LTB₄initiated a marked decrement in PSDP (FIG. 1) with a time course of PIPPremodeling concurrent with LTB₄ kinetics of cellular activation(Borgeat, P., and Naccache, P. H. (1990) Biosynthesis and biologicalactivity of leukotriene B₄ . Clin. Biochem. 23, 459-468; Levy, B. D.,Petasis, N. A., and Serhan, C. N. (1997) Polyisoprenyl phosphates inintracellular signalling. Nature 389, 985-989).

[0235] 15-epimer LX Analog Switches the LTB₄ Program to Enhance PSDP.

[0236] Both LXA₄ and some 15-epi-LXA₄ stable analogs act at the LXA₄receptor on PMN, inhibiting infiltration in vivo. To determine if LX and15-epi-LX mediate inhibitory actions via PIPP signaling, the impact of a15-epi-LXA₄ analog (15-epi-LXa) (100 nM, 5 min, 37° C.) on LTB₄ (100nM)-stimulated changes in PSDP was examined using [³²P] labeling of PMNlipids (vide supra, in parallel incubations). Alone, 15-epi-LXa did notaffect the rate of PIPP remodeling (FIG. 1B). Of interest, exposure toLTB₄ in the presence of equimolar 15-epi-LXa not only prevented the LTB₄initiated decrease in PSDP, but additionally stimulated a significantincrease (˜72%) in [³²P]-PSDP at 60 sec (FIG. 1C). PSDP levels continuedto rise for at least 300 sec after exposure to LTB₄ (FIG. 1B). NativeLXA₄ and its related LXA₄ receptor agonist, 16-phenoxy-LXA₄-methylester, gave qualitatively similar responses as 15-epi-LXa with a rankorder of potency of 15-epi-LXa>16-phenoxy-LXA₄>LXA₄ with 15-epi-LXa 1-2orders of magnitude more potent. These results indicate that 15-epi-LXa,which inhibits LTB₄ responses in vivo (Takano, T., Fiore, S., Maddox, J.F., Brady, H. R., Petasis, N. A., and Serhan, C. N. (1997)Aspirin-triggered 15-epi-lipoxin A₄ (LXA₄) and LXA₄ Stable analogues arepotent inhibitors of acute inflammation: Evidence for anti-inflammatoryreceptors. J. Exp. Med. 185, 1693-1704), dramatically switchesLTB₄-initiated PIPP signaling. Moreover, increases in PSDP levels evokedby coactivation of the LXA₄ and LTB₄ receptors indicate that the timecourse of PSDP accumulation correlated with regulation of LTB₄'s actionsby LX and 15-epi-LXa (vide infra).

[0237] 15-epi-LXa Inhibits LTB₄-stimulated PLD Activity and O₂Generation.

[0238] LTB₄-stimulated PLD activity is associated with morphologicchange, degranulation and O₂ ⁻ production in PMN (Olson, S. C., andLambeth, J. D. (1996) Biochemistry and cell biology of phospholipase Din human neutrophils. Chem. Phys. Lipids 80, 3-19; Zhou, H. -L.,Chabot-Fletcher, M., Foley, J. J., Sarau, H. M., Tzimas, M. N., Winkler,J. D., and Torphy, T. J. (1993) Association between leukotrieneB₄-induced phospholipase D activation and degranulation of humanneutrophils. Biochem. Pharmacol. 46, 139-148). To determine whether LTand LX-mediated remodeling of PIPP correlates with specific cellsignaling events, PLD activity was monitored in cell lysates from thesame incubations used in FIG. 1. LTB₄ gave increases in PLD activitythat were maximal by 60 sec (FIG. 2A). These values for LTB₄ and PLD areconsistent with those of earlier reports (Gomez-Cambronero, J. (1995)Immunoprecipitation of a phospholipase D activity withantiphosphotyrosine antibodies. J. Interferon Cytokine Res. 15, 877-885;Zhou, H. -L., Chabot-Fletcher, M., Foley, J. J., Sarau, H. M., Tzimas,M. N., Winkler, J. D., and Torphy, T. J. (1993) Association betweenleukotriene B₄-induced phospholipase D activation and degranulation ofhuman neutrophils. Biochem. Pharmacol. 46, 139-148). In the presence of15-epi-LXa, LTB₄-stimulated PLD activity was inhibited (˜81%) at 60 sec(FIG. 2A&B). 15-epi-LXa also potently inhibited LTB₄-stimulated O₂ ⁻generation (FIG. 2C). Together, these findings indicate thatligand-receptor interaction that signals opposing cellular responsesgave an inverse relationship between [³²P]-PSDP levels and PLD activity,demonstrating that PSDP could regulate PLD.

[0239] Direct Inhibition of both Plant and Mammalian PLD.

[0240] To determine whether polyisoprenyl phosphates act directly onPLD, PSDP and closely related lipids were incubated with purified plantenzyme (EC 3.1.4.4; Vm=0.29 mmoles/sec, Km=1.4 mM). As seen in FIG. 3,PSDP inhibited cPLD in a concentration-dependent fashion (10 to 1000 nM)with a Ki of 20 nM ((PSDP) =10 nM). Lineweaver-burk analyses (FIG. 3)were consistent with a competitive inhibition model. Closely relatedlipids, such as PSMP (minus only one phosphate), showed a greater than100-fold loss in inhibitory potency compared to PSDP (Table I).Comparable inhibition was not evident with other polyisoprenoids (i.e.,FDP and squalene) or a PLD product (PA). It was determined whether PSDPcould also inhibit mammalian PLD by determining recombinant human PLD1bkinetics in vitro with PSDP. The recombinant enzyme (Vm=0.36 nmoles/sec,Km=13.8 mM) was also dramatically inhibited by PSDP with a Ki of 6 nM(Table I). TABLE 1 PSDP selectively inhibits phospholipase D: structureactivity relationship with related endogenous lipids^(a) Lipid EnzymeK_(m) (mM) V_(m) cPLD 1.4 0.29 rhPLD1b 13.8  0.36 K_(m app) (mM)V_(m app) K_(i) (nM) Presqualene diphosphate cPLD 2.1 0.25 20  rhPLD1b3.1 0.03 6 Presqualene cPLD 3.1 0.36 3210   monophosphate Squalene cPLD4.0 0.46 0 Farnesyl diphosphate cPLD 0.9 0.25 0 Phosphatidic acid cPLD3.6 0.43 0

[0241] Because PLD activation occurs in vivo in the presence of manycofactors which modulate its activity, it was also determined the impactof PSDP on PLD activity in PMN lysates. Sixty seconds following LTB₄,PSDP levels decreased (28%, FIG. 1) and PLD activity was maximal (FIG.2). Addition of PSDP (100 nM) to PMN lysates at this time (60 sec, LTB₄100 nM) gave 89.5 +/− 9.7% inhibition of PLD activity. Collectively,these results indicated that PSDP was a potent inhibitor of both plantand mammalian PLD's and establish a critical role for both the terminalphosphate and the isoprenoid chain length in PSDP's action with PLDactivity.

[0242] The present results characterize PIPP remodeling as a rapidswitch for “stop” signaling used by an extracellular regulator of PMNresponses. LTB₄ receptor activation initiated a rapid and transientdecrease in PSDP (FIG. 1) that coincided temporally with increased PLDactivity (FIG. 2). As PSDP remodeling returned toward baseline values,PLD activity decreased, revealing an inverse relationship and suggestinga role for PSDP in the regulation of this pivotal lipid-modifyingenzyme. Cells exposed to LTB₄ and an LXA₄ receptor agonist (15-epi-LXa)showed a dramatic switch in PSDP remodeling to give increased [³²P]-PSDPand marked inhibition of both PLD activity and superoxide aniongeneration (FIGS. 1 & 2). In addition, synthetic PSDP was a selectiveand potent inhibitor of isolated PLD (FIG. 3, Table I), a property notshared by other closely related lipids. Taken together, the reciprocalrelationship between PSDP levels and PLD activity as well as directinhibition of recombinant human PLD1 b, purified cPLD and PLD activityin PMN lysates support a role for PSDP as an endogenous lipid regulatorof PMN PLD activity. The different temporal profiles of PIPP remodelinginitiated upon receptor activation by PMN ligands with opposing actions(i.e., stimulation and inhibition) suggest that PIPP remodeling and PSDPitself may serve as important components in intracellular signaling, inparticular as “stop” signals.

[0243] Cholesterol is not a biosynthetic product in PMN, as they lack amixed function oxidase and cyclase necessary for its endogenousformation from acetate (Shechter, I., Fogelman, A. M., and Popjak, G.(1980) A deficiency of mixed function oxidase activities in thecholesterol biosynthetic pathway of human granulocytes. J. Lipid Res.21, 277-283). In view of the present findings, the resultantbiosynthetic termination at squalene in PMN suggests that products suchas squalene's direct precursor, PSDP, carries functions distinct fromcholesterol biosynthesis. Hence, it is likely that the PIPP signalingpathway uncovered in human PMN may extend to other cell types. Inaddition to dietary influences known to impact mevalonate andpolyisoprenyl phosphate biosynthesis, PSDP formation is also activelyregulated by soluble immune stimuli and growth factors (FIG. 1B, C).Granulocyte/macrophage-colony stimulating factor, for example, increasesPSDP remodeling in PMN whereas the chemotactic peptide, fMLP, triggers(within seconds) rapid decrements in PSDP and reciprocal increments inPSMP that return to baseline within 5-10 minutes (Levy, B. D., Petasis,N. A., and Serhan, C. N. (1997) Polyisoprenyl phosphates inintracellular signalling. Nature 389, 985-989). This time course of PIPPremodeling is similar in magnitude and extent to LTB₄-initiateddecrements in PSDP (FIG. 1B & C) and correlates well with the timecourse of activating neutrophil responses such as O₂ ⁻ generation, whichis inhibited by PSDP. The presence of PSDP in peripheral blood PMNdespite their inability to generate cholesterol from endogenous sources,its rapid remodeling in response to receptor-mediated inflammatorystimuli of diverse classes of receptor agonist, and its ability toinhibit PLD activity and NADPH oxidase at nanomolar levels aresupportive evidence for a role for PSDP as a novel negativeintracellular signal. Thus, this newly uncovered PIPP signaling mightfunction to decrease negative signal levels, in contrast to thewell-appreciated phosphotidylinositol signaling pathways (reviewed inPettit, T. R., Martin, A., Horton, T., Liossis, C., Lord, J. M., andWakelam, M. J. O. (1997) Diacylglycerol and phosphatidate generated byphospholipases C and D, respectively, have distinct fatty acidcompositions and functions. J. Biol. Chem. 272, 17354-17359) that, whenactivated, rapidly generate positive intracellular stimuli (e.g.,inositol trisphosphate, diacylglycerol & Ca²⁺).

[0244] Aspirin, the lead non-steroidal anti-inflammatory drug, alsoeffects cholesterol biosynthesis by mechanisms that remain to becompletely elucidated (Rabinowitz, J. L., Baker, D. G., Villanueva, T.G., Asanza, A. P., and Capuzzi, D. M. (1992) Liver lipid profiles ofadults taking therapeutic doses of aspirin. Lipids 27, 311-314). Beyondits well-appreciated inhibition of cyclooxygenase (COX), aspirin canpirate this system to set in place an anti-inflammatory circuitgenerating 15-epi-LX, carbon 15-R-epimers of the natural15-S-containing-LX, during cell-cell interactions by aspirin-acetylatedCOX-2 and 5-lipoxygenase (FIG. 1A and Chiang, N., Takano, T., Clish, C.B., Petasis, N. A., Tai, H.-H., and Serhan, C. N. (1998)Aspirin-triggered 15-epi-lipoxin A₄ (ATL) generation by human leukocytesand murine peritonitis exudates: development of a specific 15-epi-LXA₄ELISA. J. Pharmacol Exper. Ther. 287, 779-790). These aspirin-triggeredLX carry anti-inflammatory and anti-proliferative properties (Claria,J., and Serhan, C. N. (1995) Aspirin triggers previously undescribedbioactive eicosanoids by human endothelial cell-leukocyte interactions.Proc. Natl. Acad. Sci. 92, 9475-9479; Serhan, C. N. (1997) Lipoxins andNovel Aspirin-Triggered 15-epi-Lipoxins: A Jungle of Cell-CellInteractions or a Therapeutic Opportunity? Prostaglandins 53, 107-137)and may mediate a component of aspirin's beneficial therapeutic actions.As observed in the present experiments, LXA₄ receptor activation by a15-epi-LX mimetic reversed PSDP remodeling initiated by LTB₄ receptors,leading to increases in PSDP levels (FIG. 1). Since the 15-epi-LXainhibited both PLD activity and superoxide anion generation (FIG. 2),these results implicate PIPP remodeling as a component of the cellularbasis for aspirin's inhibition of excessive inflammatory responses. Inaddition to regulating LTB₄'s stimulatory actions, this novel mechanismof inhibition of LTB₄ receptor signaling may also play broader roles inhost defense, as this receptor was recently identified as a co-receptorfor HIV-1 (Owman, C., Garzino-Demo, A., Cocchi, F., Popovic, M.,Sabirsh, A., and Gallo, R. (1998) The leukotriene B₄ receptor functionsas a novel type of coreceptor mediating entry of primary HIV-1 isolatesinto CD4-positive cells. Proc. Natl. Acad. Sci. 95, 9530-9534).

[0245] Hydrolysis of PC to PA by PLD appears crucial in transmembranesignaling by a wide range of receptor classes during PMN activation(Olson, S. C., and Lambeth, J. D. (1996) Biochemistry and cell biologyof phospholipase D in human neutrophils. Chem. Phys. Lipids 80, 3-19).Both G-protein linked receptors and receptor tyrosine kinases activatePLD. In leukocytes, several factors including PKCα (in akinase-independent manner) and increased intracellular calcium canactivate PLD1 (Exton, J. H. (1997) New developments in phospholipase D.J. Biol. Chem. 272, 15579-15582). FMLP-stimulated PLD activity in PMN isincreased by membrane association of the ADP-ribosylation factor (ARF)and small GTPase RhoA (Fensome, A., Whatmore, J., Morgan, C., Jones, D.,and Cockcroft, S. (1998) ADP-ribosylation factor and Rho proteinsmediate fMLP-dependent activation of phospholipase D in humanneutrophils. J. Biol. Chem. 273, 13157-13164). Of considerable interesthere, PSDP directly inhibited recombinant hPLD1b in the absence ofregulatory proteins (see Table I). These results suggest that PSDP mayinhibit PLD at its catalytic center and is likely to act at other PLDisoforms, such as PLD1a and PLD2 isoforms where the catalytic centersare conserved. PSDP's ability to serve as an endogenous inhibitor of PLDlikely results from PSDP's unique three-dimensional and physicalchemical properties which might now serve as a template for thepreparation of more potent PLD inhibitors by design to fulfill thestructure activity relationship uncovered here.

[0246] Regulation of PMN activation in complex host responses iscontrolled in part by soluble mediators and, in particular, by autacoidswith opposing actions (Serhan, C. N., Haeggstrom, J. Z., and Leslie, C.C. (1996) Lipid mediator networks in cell signaling: update and impactof cytokines. FASEB J. 10, 1147-1158), such as LT and LX, that here gavemarkedly different profiles for PIPP remodeling (FIG. 1). In most celltypes, PSDP is appreciated as a biosynthetic intermediate in cholesterolproduction by microsomal squalene synthase, which catalyzes head-to-headcondensation of two FDP (Jarstfer, M. B., Blagg, B. S. J., Rogers, D.H., and Poulter, C. D. (1996) Biosynthesis of squalene. Evidence for atertiary cyclopropylcarbinyl cationic intermediate in the rearrangementof presqualene diphosphate to squalene. J. Amer. Chem. Soc. 118,13089-13090). Ligand-operated rapid remodeling of PSDP in PMN is likelyto occur in membranes in proximity to LTB₄ and LXA₄ receptors andsuggests a non-microsomal pool of PSDP that may result from 1) novelbiosynthetic and/or metabolic pathways or 2) intracellular traffickingof PIPP with proteins from endoplasmic reticulum to membrane domains.Incorporation of [³²P] from ATP into PSDP but not FDP (see Results) isfurther evidence in support of a novel route for PSDP formation in PMN.The present results suggest that PIPP remodeling is linked to cellsurface receptor activation and is involved in the intracellulartransmission of extracellular ligands with opposing biological actions.In the present working model, a “negative lipid signal” (i.e., PSDP) isheld at a set point, like a ratchet, in “resting” cells. Incomingpositive signals (LTB₄, fMLP, etc.) initiate the degradation andinactivation of this inhibitory lipid (e.g., remodeling PSDP to theinactive monophosphate species, PSMP) (FIG. 1A and ref 22). Thus, PIPPremodeling enables mounting of intracellular positive signals thatthreshold for activation of select cellular processes. This type ofsignaling may explain the selectivity and tight coupling required byagonists such as LTB₄ that stimulate highly specialized functionalresponses of PMN such as chemotaxis, granule mobilization and superoxideanion generation. The extent to which this model of cell signaling,namely receptor-initiated degradation of negative lipid signals, occurswith other receptors and cell types remains for further studies.

[0247] In summary, ligand-operated rapid remodeling of PIPPs in humanPMN and direct inhibition of PLD activity at nanomolar levels support arole for PSDP as an intracellular signal and provide novel intracellulartargets by which PSDP can regulate cellular responses (Levy, B. D.,Petasis, N. A., and Serhan, C. N. (1997) Polyisoprenyl phosphates inintracellular signalling. Nature 389, 985-989). Given the wideoccurrence of PIPP and critical role of PLD in the plant and animalkingdoms, PIPP remodeling and direct inhibition of PLD first establishedhere in human PMN may have wider implications in cell signaling in othercell types and species (Martin, A., Saqib, K. M., Hodgkin, M. N., Brown,F. D., Pettit, T. R., Armstrong, S., and Wakelam, M. J. O. (1997) Roleand regulation of phospholipase D signalling. Biochem. Soc. Trans. 25,1157-1160; Bach, T. J. (1995) Some new aspects of isoprenoidbiosynthesis in plants—a review. Lipids 30, 191-202). The presentresults are the first to show direct inhibition of a phospholipaseinvolved in signal transduction by an endogenous intracellular lipid andset forth a new paradigm for lipid-protein interactions in the controlof cellular responses, namely receptor-initiated degradation of arepressor lipid, that is also subject to regulation by aspirin ingestionvia the actions of aspirin-triggered 15-epimer LX. Together, theseresults suggest that PIPP signaling pathways might also be of interestin pharmacologic interventions and specifically that the conformation ofPSDP can serve as a template for design of novel inhibitors.

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[0285] One having ordinary skill in the art will appreciate furtherfeatures and advantages of the invention based on the above-describedembodiments. Accordingly, the invention is not to be limited by what hasbeen particularly shown and described, except as indicated by theappended claims. All publications and references cited herein, includingthose in the background section, are expressly incorporated herein byreference in their entirety.

What is claimed is:
 1. A method for modulating a disease or conditionassociated with phospholipase D (PLD) initiated polymorphoneutrophil(PMN) inflammation in a subject, comprising administering to the subjectan effective anti-inflammatory amount of a lipoxin analog having theformula

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl; (vii) a detectablelabel molecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN), provided whenQ₁ is CN, then X is absent; wherein Q₃ and Q₄ are each independently O,S or NH; wherein one of R₂ and R₃ is a hydrogen atom and the other is(a) H; (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched; (c) a cycloalkyl of 3 to 6 carbon atoms,inclusive; (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which maybe straight chain or branched; or (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or—S—; wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, whichmay be straight chain or branched and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which may be straight chain or branched,provided when R_(b) is 0, then R_(b) is a hydrogen atom; wherein R₄ is(a) H; (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched; wherein R₅ is

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group; wherein Y₁ is —OH,methyl, —SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chainor branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH_(a)Z_(b)where a+b=3, a=0 to 3, b=0 to 3 and Z is cyano, nitro or a halogen;wherein R₆ is (a) H; (b) an alkyl from 1 to 4 carbon atoms, inclusive,straight chain or branched; wherein T is O or S, and pharmaceuticallyacceptable salts thereof, such that a disease or condition associatedwith PLD initiated polymorphoneutrophil (PMN) inflammation in a subjectis modulated.
 2. The method of claim 1, wherein said method is performedin vitro.
 3. The method of claim 1, wherein said method is performed invivo.
 4. A method for treating phospholipase D (PLD) initiatedpolymorphoneutrophil (PMN) inflammation in a subject, comprisingadministering to the subject an effective anti-inflammatory amount of alipoxin analog having the formula

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl; (vii) a detectable labelmolecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN), provided whenQ₁ is CN, then X is absent; wherein Q₃ and Q₄ are each independently O,S or NH; wherein one of R₂ and R₃ is a hydrogen atom and the other is(a) H; (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched; (c) a cycloalkyl of 3 to 6 carbon atoms,inclusive; (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which maybe straight chain or branched; or (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or—S—; wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, whichmay be straight chain or branched and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which may be straight chain or branched,provided when R_(b) is 0, then R_(b) is a hydrogen atom; wherein R₄ is(a) H; (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched; wherein R₅ is

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group; wherein Y₁ is —OH,methyl, —SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chainor branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH_(a)Z_(b)where a+b=3, a=0 to 3, b=0 to 3 and Z is cyano, nitro or a halogen;wherein R₆ is (a) H; (b) an alkyl from 1 to 4 carbon atoms, inclusive,straight chain or branched; wherein T is O or S, and pharmaceuticallyacceptable salts thereof, such that PLD initiated polymorphoneutrophil(PMN) inflammation is treated in a subject.
 5. The method of claim 1,wherein said method is performed in vitro.
 6. The method of claim 1,wherein said method is performed in vivo.
 7. A method for modulating adisease or condition associated with phosphlipase D (PLD) initiatedsuperoxide generation or degranulation activity in a subject, comprisingadministering to the subject an effective anti-PLD amount of a lipoxinanalog having the formula

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl; (vii) a detectable labelmolecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN), provided whenQ₁ is CN, then X is absent; wherein Q₃ and Q₄ are each independently O,S or NH; wherein one of R₂ and R₃ is a hydrogen atom and the other is(a) H; (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched; (c) a cycloalkyl of 3 to 6 carbon atoms,inclusive; (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which maybe straight chain or branched; or (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or—S—; wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, whichmay be straight chain or branched and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which may be straight chain or branched,provided when R_(b) is 0, then R_(b) is a hydrogen atom; wherein R₄ is(a) H; (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched; wherein R₅ is

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group; wherein Y₁ is —OH,methyl, —SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chainor branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH_(a)Z_(b)where a+b=3, a=0 to 3, b=0 to 3 and Z is cyano, nitro or a halogen;wherein R₆ is (a) H; (b) an alkyl from 1 to 4 carbon atoms, inclusive,straight chain or branched; wherein T is O or S, and pharmaceuticallyacceptable salts thereof, such that a disease or condition associatedwith PLD initiated superoxide generation or degranulation activity in asubject is modulated.
 8. The method of claim 7, wherein said method isperformed in vitro.
 9. The method of claim 7, wherein said method isperformed in vivo.
 10. A method for treating phospholipase D (PLD)initiated superoxide generation or degranulation in a subject,comprising administering to the subject an effective anti-PLD amount ofa lipoxin analog having the formula

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl; (vii) a detectable labelmolecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN), provided whenQ₁ is CN, then X is absent; wherein Q₃ and Q₄ are each independently O,S or NH; wherein one of R₂ and R₃ is a hydrogen atom and the other is(a) H; (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched; (c) a cycloalkyl of 3 to 6 carbon atoms,inclusive; (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which maybe straight chain or branched; or (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or—S—; wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, whichmay be straight chain or branched and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which may be straight chain or branched,provided when R_(b) is 0, then R_(b) is a hydrogen atom; wherein R₄ is(a) H; (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched; wherein R₅ is

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group; wherein Y₁ is —OH,methyl, —SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chainor branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH_(a)Z_(b)where a+b 3, a=0 to 3, b=0 to 3 and Z is cyano, nitro or a halogen;wherein R₆ is (a) H; (b) an alkyl from 1 to 4 carbon atoms, inclusive,straight chain or branched; wherein T is O or S, and pharmaceuticallyacceptable salts thereof, such that PLD initiated superoxide generationor granulation is treated in a subject.
 11. The method of claim 10,wherein said method is performed in vitro.
 12. The method of claim 10,wherein said method is performed in vivo.
 13. A packaged pharmaceuticalcomposition for treating a disease or condition associated withphospholipase D (PLD) initiated activity in a subject, comprising: acontainer holding a therapeutically effective amount of at least onelipoxin compound having the formula

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl; (vii) a detectable labelmolecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN), provided whenQ₁ is CN, then X is absent; wherein Q₃ and Q₄ are each independently O,S or NH; wherein one of R₂ and R₃ is a hydrogen atom and the other is(a) H; (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched; (c) a cycloalkyl of 3 to 6 carbon atoms,inclusive; (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which maybe straight chain or branched; or (e) R^(a)Q₂R_(b) wherein Q₂ is —O— or—S—; wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, whichmay be straight chain or branched and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which may be straight chain or branched,provided when R_(b) is 0, then R_(b) is a hydrogen atom; wherein R₄ is(a) H; (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched; wherein R₅ is

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁ wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group; wherein Y₁ is —OH,methyl, —SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chainor branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH_(a)Z_(b)where a+b=3, a=0 to 3, b=0 to 3 and Z is cyano, nitro or a halogen;wherein R₆ is (a) H; (b) an alkyl from 1 to 4 carbon atoms, inclusive,straight chain or branched; wherein T is O or S, and pharmaceuticallyacceptable salts thereof; and instructions for using said lipoxincompound for treating a disease or condition associated with PLDinitiated activity in the subject.
 14. A packaged pharmaceuticalcomposition for treating phospholipase D initiated activity in asubject, comprising: a container holding a therapeutically effectiveamount of at least one lipoxin compound having the formula

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl; (vii) a detectable labelmolecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN), provided whenQ₁ is CN, then X is absent; wherein Q₃ and Q₄ are each independently O,S or NH; wherein one of R₂ and R₃ is a hydrogen atom and the other is(a) H; (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched; (c) a cycloalkyl of 3 to 6 carbon atoms,inclusive; (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which maybe straight chain or branched; or (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or—S—; wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, whichmay be straight chain or branched and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which may be straight chain or branched,provided when R_(b) is 0, then R_(b) is a hydrogen atom; wherein R₄ is(a) H; (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched; wherein R₅ is

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group; wherein Y₁ is —OH,methyl, —SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chainor branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH_(a)Z_(b)where a+b=3, a=0 to 3, b=0 to 3 and Z is cyano, nitro or a halogen;wherein R₆ is (a) H; (b) an alkyl from 1 to 4 carbon atoms, inclusive,straight chain or branched; wherein T is O or S, and pharmaceuticallyacceptable salts thereof; and instructions for using said lipoxincompound for treating PLD initiated activity in the subject.
 15. Apackaged pharmaceutical composition for treating a disease or conditionassociated with phospholipase D (PLD) initiated superoxide generation ordegranulation activity in a subject, comprising: a container holding atherapeutically effective amount of at least one lipoxin compound havingthe formula

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl; (vii) a detectable labelmolecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN), provided whenQ₁ is CN, then X is absent; wherein Q₃ and Q₄ are each independently O,S or NH; wherein one of R₂ and R₃ is a hydrogen atom and the other is(a) H; (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched; (c) a cycloalkyl of 3 to 6 carbon atoms,inclusive; (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which maybe straight chain or branched; or (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or—S—; wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, whichmay be straight chain or branched and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which may be straight chain or branched,provided when R_(b) is 0, then R_(b) is a hydrogen atom; wherein R₄ is(a) H; (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched; wherein R₅ is

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group; wherein Y₁ is —OH,methyl, —SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chainor branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH_(a)Z_(b)where a+b=3, a=0 to 3, b=0 to 3 and Z is cyano, nitro or a halogen;wherein R₆ is (a) H; (b) an alkyl from 1 to 4 carbon atoms, inclusive,straight chain or branched; wherein T is O or S, and pharmaceuticallyacceptable salts thereof; and instructions for using said lipoxincompound for treating a disease or condition associated with PLDinitiated superoxide generation or degranulation activity in thesubject.
 16. A packaged pharmaceutical composition for treatingphospholipase D (PLD) initiated superoxide generation or degranulationactivity in a subject, comprising: a container holding a therapeuticallyeffective amount of at least one lipoxin compound having the formula

wherein X is R₁, OR₁, or SR₁; wherein R₁ is (i) a hydrogen atom; (ii) analkyl of 1 to 8 carbons atoms, inclusive, which may be straight chain orbranched; (iii) a cycloalkyl of 3 to 10 carbon atoms; (iv) an aralkyl of7 to 12 carbon atoms; (v) phenyl; (vi) substituted phenyl

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO_(2,)—CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR₁, wherein R_(x) is 1 to 8 carbon atoms, inclusive, which maybe a straight chain or branched, and hydroxyl; (vii) a detectable labelmolecule; or (viii) a straight or branched chain alkenyl of 2 to 8carbon atoms, inclusive; wherein Q₁ is (C═O), SO₂ or (CN), provided whenQ₁ is CN, then X is absent; wherein Q₃ and Q₄ are each independently O,S or NH; wherein one of R₂ and R₃ is a hydrogen atom and the other is(a) H; (b) an alkyl of 1 to 8 carbon atoms, inclusive, which may be astraight chain or branched; (c) a cycloalkyl of 3 to 6 carbon atoms,inclusive; (d) an alkenyl of 2 to 8 carbon atoms, inclusive, which maybe straight chain or branched; or (e) R_(a)Q₂R_(b) wherein Q₂ is —O— or—S—; wherein R_(a) is alkylene of 0 to 6 carbons atoms, inclusive, whichmay be straight chain or branched and wherein R_(b) is alkyl of 0 to 8carbon atoms, inclusive, which may be straight chain or branched,provided when R_(b) is 0, then R_(b) is a hydrogen atom; wherein R₄ is(a) H; (b) an alkyl of 1 to 6 carbon atoms, inclusive, which may be astraight chain or branched; wherein R₅ is

 wherein Z_(i) Z_(ii), Z_(iii), Z_(iv) and Z_(v) are each independentlyselected from —NO₂, —CN, —C(═O)—R₁, —SO₃H, a hydrogen atom, halogen,methyl, —OR_(x), wherein R_(x) is 1 to 8 carbon atoms, inclusive, whichmay be a straight chain or branched, and hydroxyl or a substituted orunsubstituted, branched or unbranched alkyl group; wherein Y₁ is —OH,methyl, —SH, an alkyl of 2 to 4 carbon atoms, inclusive, straight chainor branched, an alkoxy of 1 to 4 carbon atoms, inclusive, or CH_(a)Z_(b)where a+b=3, a=0 to 3, b 0 to 3 and Z is cyano, nitro or a halogen;wherein R₆ is (a) H; (b) an alkyl from 1 to 4 carbon atoms, inclusive,straight chain or branched; wherein T is O or S, and pharmaceuticallyacceptable salts thereof; and instructions for using said lipoxincompound for treating PLD initiated superoxide generation ordegranulation activity in the subject.